Mycoplasma penetrans is a bacterium associated with immunocompromised patients, and has been implicated as a potential cofactor in AIDS progression. Although exactly how it harms host cells is unclear, like many other pathogenic mycoplasmas it attaches to host cells by a polarized appendage, the attachment organelle (AO), which is also the leading end of the cell during gliding motility. Interference with AO-mediated processes, including its assembly, adherence, and motility, is a potential target for the development of therapeutic agents that could relieve infected patients, which include a substantial number of HIV-positive individuals. Furthermore, understanding how mycoplasmas, which have served as models for minimalistic cells, generate cell polarity, will be generally informative to cell biologists attempting to understand generation of cell polarity in more complex organisms, ranging from other prokaryotes to human cells. Comparison of the M. penetrans genome with more distantly related mycoplasma species like Mycoplasma pneumoniae and Mycoplasma mobile reveals the absence of homologs of AO proteins of these species. The M. penetrans AO has distinct ultrastructural features relative to these other species, including those of its underlying cytoskeletal elements. Furthermore, unlike M. mobile, M. penetrans motility does not directly depend on ATP hydrolysis. All these data indicate that the M. penetrans AO is only superficially convergent with analogous structures in other species, and therefore understanding the molecular basis for M. penetrans AO assembly and function cannot be inferred from information from these species. On the other hand, Mycoplasma iowae, a close relative of M. penetrans, has an ultrastructurally similar AO, and its genome contains homologs of proteins we have preliminarily identified as M. penetrans AO proteins, including a series of cytoskeletal proteins and a candidate adhesin, P42. Although biochemical and cell biological studies have begun to reveal the components and mechanisms associated with the M. penetrans AO, and we will continue to employ them, the absence of a genetic system in this organism has significantly slowed progress toward solving these problems. However, our preliminary results indicate that unlike M. penetrans, M. iowae is capable of being transformed with Tn4001- derived transposons, with tetracycline resistance available as a selectable marker. In this proposal we describe experiments aimed at developing M. iowae as a genetic system to model the M. penetrans AO, studying the localization of a green fluorescent protein fusion to a preliminarily identified AO component as well as generating a null mutant in the gene for that protein. We will also investigate the role of P42 in M. penetran adherence and motility through antibody inhibition studies. Finally, we will use biochemical fractionation and mass spectrometry to identify other proteins of the M. penetrans AO. Finally, this AREA proposal renewal will also accomplish the training of undergraduate and graduate students at Miami University.